甘蔗CBL-CIPK基因家族的鉴定和表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0830

摘要/Abstract

摘要：

【目的】甘蔗是重要的糖料作物，温度、盐碱、水分等因素是制约其生长发育的关键环境因素。类钙调磷酸酶B蛋白CBL（calcineurin B-like protein）是一类Ca²⁺结合蛋白，通过与其特定的蛋白激酶CIPK（CBL-interacting protein kinase）作用，在Ca²⁺信号传导通路，尤其是逆境信号传导通路中发挥重要作用。目前，甘蔗全基因组测序已完成，但其CBL-CIPK基因家族成员尚未确定，互作调控机理依然未知。本研究确定了甘蔗CBL、CIPK成员并揭示了CBL-CIPK互作关系，为研究甘蔗CBL-CIPK的互作机理提供基因资源和理论基础。【方法】以甘蔗品种‘GT58’为材料，通过RT-qPCR技术分析CBLs、CIPKs在低温、高温、NaHCO₃和PEG处理等4种非生物胁迫下的表达水平，利用酵母双杂交试验分析SsCBLs和SsCIPKs之间的相互作用。【结果】甘蔗全基因组中共有19个CBL基因和82个CIPK基因，分布在不同的进化分支且存在基因复制现象，基因家族成员之间理化性质差异较大，结构域与蛋白基序具有高度保守性，顺式作用元件分布多样；转录水平上，SsCBL7/SsCBL12/SsCIPK1/SsCIPK5的表达水平易受低温、高温、干旱和高盐等多种非生物胁迫的调控；蛋白水平上，SsCBL1与SsCIPK47和SsCIPK81相互作用，SsCBL8与SsCIPK47和SsCIPK81相互作用。【结论】CBL-CIPK互作网络可能在甘蔗生长发育过程中响应非生物胁迫，发挥重要作用。

关键词: 甘蔗, SsCBL, SsCIPK, 非生物胁迫, 酵母双杂交

Abstract:

【Objective】 Sugarcane is an important sugar crop, and temperature, salinity, water and other factors are the key environmental factors restricting its growth and development. Calcineurin B-Like protein（CBL）family represents a group of Ca²⁺ binding proteins and plays a key role in decoding calcium signals by specifically interacting with and regulating a family of protein kinases CIPKs（for CBL-interacting protein kinases）in abiotic stress signal transduction pathway. At present, the whole genome of sugarcane has been sequenced, but its CPL-CIPK gene family members have not been identified, and the interaction regulation mechanism is still unknown. Here, the members of CBL and CIPK in sugarcane were identified and the CPL-CIPK interaction relationship was revealed, which may provide gene resources and theoretical basis for studying the mechanism of CPL-CIPK interaction in sugarcane.【Method】 The expression levels of CBLs and CIPKs under low temperature, high temperature, NaHCO₃ and PEG were analyzed by RT-qPCR from sugarcane GT58. The interactions between SsCBLs and SsCIPKs were analyzed by yeast two-hybrid assay.【Result】 There were 19 CBL genes and 82 CIPK genes in the whole genome of sugarcane, which distributed in different evolutionary branches with gene replication. The physicochemical properties of gene family members were different, the structural domains and protein motifs were highly conserved, and the distribution of cis-acting elements was diverse. SsCBL7/SsCBL12/SsCIPK1/SsCIPK5 were specifically expressed under low temperature, high temperature, drought and high salt and other abiotic stress regulation. Meanwhile, SsCBL1 interacted with SsCIPK47 and SsCIPK81, and SsCBL8 interacted with SsCIPK47 and SsCIPK81.【Conclusion】 The CBL-CIPK signaling system may play an important role in response to abiotic stress during the growth and development of sugarcane.

Key words: sugarcane, SsCBL, SsCIPK, abiotic stresses, yeast two-hybrid

辛奇, 李压凡, 尹铮, 张晓丹, 陈霆, 刘晓华. 甘蔗CBL-CIPK基因家族的鉴定和表达分析[J]. 生物技术通报, 2024, 40(2): 197-211.

XIN Qi, LI Ya-fan, YIN Zheng, ZHANG Xiao-dan, CHEN Ting, LIU Xiao-hua. Identification and Expression Analysis of the CBL-CIPK Gene Family in Sugarcane[J]. Biotechnology Bulletin, 2024, 40(2): 197-211.

图/表 9

表1 甘蔗19个CBLs的理化性质统计表

Table 1 Statistical table of physico-chemical properties of 19 CBLs of sugarcane

基因 Gene	基因ID Gene ID	基因长度 Gene length /bp	正负链 Strand	mRNA长度 mRNA length/bp	氨基酸数目 Amino acid/aa	等电点 pI	外显子个数 Exon number	染色体定位 Chromosome	相对分子质量 Molecular weight/kD	亚细胞定位预测 Subcellular localization
SsCBL1	Sspon.01G0014730-1A	3 124	＋	579	193	5.00	7	Chr.1A	22.26	Cyto
SsCBL2	Sspon.01G0023690-1A	3 641	＋	675	225	4.82	8	Chr.1A	25.77	Cyto
SsCBL3	Sspon.01G0014730-2B	3 431	-	639	213	4.75	8	Chr.1B	24.49	Cyto
SsCBL4	Sspon.01G0014730-3C	3 431	-	639	213	4.75	8	Chr.1C	24.49	Cyto
SsCBL5	Sspon.01G0014730-4D	3 431	-	639	213	4.75	8	Chr.1D	24.49	Cyto
SsCBL6	Sspon.01G0023690-3D	3 641	＋	675	225	4.82	8	Chr.1D	25.77	Cyto
SsCBL7	Sspon.02G0030420-1A	3 582	＋	660	220	4.92	9	Chr.2A	25.43	Chlo
SsCBL8	Sspon.02G0030420-2C	3 640	-	675	225	4.79	8	Chr.2C	25.84	Cyto
SsCBL9	Sspon.03G0011980-1A	4 077	-	738	246	6.14	10	Chr.3A	28.83	Cyto
SsCBL10	Sspon.03G0012700-1A	4 617	-	882	294	4.84	9	Chr.3A	33.11	E.R.
SsCBL11	Sspon.03G0031480-1B	4 573	-	864	288	4.84	8	Chr.3B	32.40	Chlo
SsCBL12	Sspon.03G0011980-2B	4 758	＋	882	294	5.28	10	Chr.3B	33.94	Cyto
SsCBL13	Sspon.03G0011980-1P	3 620	-	660	220	6.19	8	Chr.3B	25.66	Cyto
SsCBL14	Sspon.04G0013450-2B	3 432	＋	639	213	4.77	8	Chr.4B	24.34	Nucl
SsCBL15	Sspon.07G0003410-1A	3 363	-	633	211	4.88	8	Chr.7A	24.11	Chlo
SsCBL16	Sspon.07G0018960-1A	3 619	-	669	223	4.82	8	Chr.7A	25.68	Chlo
SsCBL17	Sspon.07G0018960-2B	3 363	-	618	206	4.99	7	Chr.7B	23.77	Chlo
SsCBL18	Sspon.07G0018960-3C	3 324	＋	612	204	4.91	7	Chr.7C	23.52	Mito
SsCBL19	Sspon.07G0018960-4D	3 485	＋	642	214	4.92	8	Chr.7D	24.69	Chlo

图1 CBL-CIPK基因家族的domain与motif分析 A：甘蔗CBL家族成员蛋白基序motif、保守结构域domain分析；B：甘蔗CIPK家族成员motif、domain分析。从左至右分别为进化关系树、motif、domain，不同颜色的模块分别代表不同的motif和domain

Fig. 1 Domain and motif analysis of CBL-CIPK gene family A: Analysis of motif, domain of sugarcane CBL family members. B: Analysis of motif, domain of sugarcane CIPK family members. From left to right are the evolutionary relationship tree, motif and domain, with different colored modules representing different motifs and domains

图2 CBL-CIPK基因家族的基因结构分析 A：甘蔗CBL家族成员基因结构；B：甘蔗CIPK家族成员基因结构；左侧为进化关系树，右侧为家族成员基因结构，绿色部分为UTR，黄色部分为CDS

Fig. 2 Analysis in the gene structures of CBL-CIPK gene family A: Gene structure of sugarcane CBL family members. B: Gene structure of sugarcane CIPK family members. Evolutionary relationship tree on the left, gene structure of family members on the right, UTR in green and CDS in yellow

图3 CBL-CIPK基因家族的染色体定位分析 A：甘蔗CBL家族成员染色体定位；B：甘蔗CIPK家族成员染色体定位

Fig. 3 Analysis for their positions on the chromosomes in CBL-CIPK gene family A: Chromosomal localization of sugarcane CBL family members. B: Chromosomal localization of sugarcane CIPK family members

图4 甘蔗CBL基因家族顺式作用元件预测统计

Fig. 4 Predicted statistics for cis-acting elements of the sugarcane CBL gene family

图5 CBL-CIPK基因家族系统发育树及共线性分析 A：多物种CBL家族系统发育进化树；B：多物种CIPK家族系统发育进化树；C：甘蔗种内CBL-CIPK基因家族共线性分析；全基因组中所有基因对均为灰色，CBL-CIPK家族成员形成基因对为红色，甘蔗为异源四倍体，1A-8D为甘蔗的32条染色体

Fig. 5 Phylogenetic tree and covariance analysis of the CBL-CIPK gene family A: Phylogenetic evolutionary tree of the multispecies CBL family. B: Phylogenetic evolutionary tree of the multispecies CIPK family. C: Analysis of intraspecific CBL-CIPK gene family covariance in sugarcane. All gene pairs in the whole genome are gray, CBL-CIPK family members form gene pairs in red, sugarcane is a heterotetraploid, and 1A-8D are the 32 chromosomes of sugarcane

图6 参与不同温度胁迫响应的SsCBLs、SsCIPKs基因的RT-qPCR分析 A：4℃处理下SsCBLs的基因表达谱；B：4℃处理下SsCIPKs的基因表达谱；C：45℃处理下SsCBLs的基因表达谱；D：45℃处理下SsCIPKs的基因表达谱

Fig. 6 RT-qPCR analysis of SsCBLs, SsCIPKs genes involved in different temperature stresses response A: Gene expression profiles of SsCBLs under 4℃ treatment. B: Gene expression profiles of SsCIPKs under 4℃ treatment. C: Gene expression profiles of SsCBLs under 45℃ treatment. D: Gene expression profiles of SsCIPKs under 45℃ treatment

图7 参与NaHCO3/PEG胁迫的SsCBLs、SsCIPKs的RT-qPCR分析 A：170 mmol/L NaHCO3处理下SsCBLs的基因表达谱；B：170 mmol/L NaHCO3处理下SsCIPKs的基因表达谱；C：150 g/L PEG-6000处理下SsCBLs的基因表达谱；D：150 g/L PEG-6000处理下SsCIPKs的基因表达谱

Fig. 7 RT-qPCR analysis of SsCBLs, SsCIPKs genes involved in NaHCO3/PEG stresses A: Gene expression profiles of SsCBLs under170 mmol/L NaHCO3 treatment. B: Gene expression profiles of SsCIPKs under 170 mmol/L NaHCO3 treatment. C: Gene expression profiles of SsCBLs under 150 g/L PEG-6000 treatment. D: Gene expression profiles of SsCIPKs under 150 g/L PEG-6000 treatment

图8 SsCBLs和SsCIPKs之间的相互作用分析

Fig. 8 Interaction between SsCBLs and SsCIPKs

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